Prostaglandins, particularly prostaglandin E2 (PGE2) are potent regulators of skeletal metabolism which can both stimulate and inhibit bone resorption and formation. PGE2 effects can be mediated by four receptors, EP1, EP2, EP3, and EP4. EP2 and EP4 increase cyclic AMP and have been implicated in stimulation of bone resorption and formation. EP1 has been implicated in mitogenesis. The first specific aim of this proposal will be to examine mice in which the EP2 receptor has been knocked out by homologous recombination. EP2 (+/+) and EP2 (-/-) mice will be studied for their response to exogenous PGE2 and for changes in bone turnover with aging. Cell and organ cultures from EP2 (+/+) and EP2 (-/-) mice will be examined for changes in osteoblast and osteoclast function in response not only to PGE2, but also to other stimulators of resorption and formation. Bone formation will be assessed in vivo by dynamic histomorphometry. DNA content, apoptosis, collagen and non-collagen protein synthesis and the expression of osteoblastic genes will be assessed both in vivo and in vitro. Bone resorption will be assessed in vivo by histologic analysis of tartrate-resistant acid phosphatase positive multi-nucleated cells (TRAP + MNC). Marrow cell cultures, spleen/osteoblast co-cultures and spleen cultures stimulated by osteoclast differentiation factor and monocyte colony stimulating factor will be examined for osteoclastogenesis by counting TRAP + MNC and pit formation on bone slices. The expression of specific genes involved in osteoclast activation will be examined in cell cultures of osteoblasts. The second specific aim will be to examine agonists and antagonists for each of the four PGE2 receptors in cell cultures of osteoblasts and in co-cultures of osteoblasts and spleen cells and in vivo. The applicants will test previously identified agents, as well as new specific agonists for all four EP1 receptors and a potent, specific, newly synthesized antagonist for the EP4 receptor. These studies will provide information on the role of EP receptors in mediating the effects of PGE2 on skeletal metabolism, and will provide models to explore the use of specific agonists and antagonists of PG receptors in the treatment of osteoporosis and other metabolic and inflammatory disorders of the skeleton.